This competitive renewal application continues to focus on discovery of novel signaling mechanisms in cardioprotection. It utilizes a nitric oxide (NO) donor induced model of cardioprotection. This model of pharmacological cardioprotection has been established in the previous funding cycle (yr 6 to yr 10). Several lines of evidence support our central hypothesis that NO acts as a multi-faceted player modulating mitochondrial signaling, rendering a protective mitochondrial phenotype, leading to cardiac myocyte survival. Recently, a role for Src-kinase is implicated in cardioprotection and several reports, in conjunction with our own preliminary data, demonstrated a mitochondrial localization of Src-kinase, a Src-kinase mediated regulation of mitochondrial electron transport complex (ETC) activity, and an increased Src-kinase activity during NO-induced cardioprotection. In view of these new developments, this completive renewal proposes to examine a novel mechanism of cardioprotection: i.e., the role of mitochondrial Src-kinase as a mandatory target of NO induced cardioprotection. The application will identify novel targets of Src-kinase in the mitochondria and it will functionally validate the novel targets as possible executors of the protective effects of Src-kinase in the mitochondria. Three specific aims are proposed. Aim 1 will interrogate Src-kinase dependent molecular mechanisms and its salutary effect on mitochondrial function following the administration of NO donors. We will determine mitochondrial Src-kinase activity, their sub-organellar localization, and their interacting partners. The necessary role of mitochondrial Src in NO cardioprotection will be examined via both pharmacological inhibition of Src-kinase and a Src-kinase knockout model. Aim 2 will fully characterize the mitochondrial proteome impacted by Src-kinase phosphorylation; it will identify alterations of the phospho-proteome during NO donor induced cardioprotection. To achieve this goal, Aim 2 will employ a new technology platform developed in house. The proposed studies will lead to the discovery of novel mitochondrial targets of Src-kinase; they will identify the basic building blocks of the mitochondrial Src-kinase pathway(s); and they will provide mechanistic insights regarding the regulatory events underlying perturbations of the Src-kinase pathway by NO donors. The feasibility of Aim 2 is supported by our exciting preliminary results; the PDH E1a (Y-157) has been shown as a novel mitochondrial target of Src-kinase. Aim 3 will examine the sufficient and necessary role of PDH as a mitochondrial target of Src-kinase, facilitating the protection of mitochondrial phenotype during NO-induced cardioprotection. Aim 3 is a logic next step of Aim 2. The proposed studies will delineate the functional consequences of Src-kinase dependent phosphorylation of the PDH E1a during NO cardioprotection in three layers: at the individual enzyme level (e.g., augmenting PDH activity), at the organelle level (e.g., preserving mitochondrial function), and at the whole organ level (e.g., reduction in myocardial infarction). We will utilize established models of isolated mitochondria (simulated ischemia), as well as a murine model of ischemic injury, to conclusively determine the role of Src-kinase modulation of PDH in cardioprotection.